There is as yet no effective vaccine to HIV despite concerted efforts by numerous groups worldwide to produce one. The target for vaccines is the envelope spike protein (gp120/gp41), which mediates binding to receptors on the surface of CD4 T cells. The problem from a vaccine perspective is that the spike protein is heavily glycosylated, with up to 25 or more N-linked glycans, creating a 'glycan shield' that prevent an immune response to the underlying protein. Yet, many HIV infected individuals develop broadly neutralizing antibodies (bnAbs) that target the dense array of glycans. Analysis these glycan- dependent bnAbs show that they bind to different regions on the spike protein, and that they recognize different types of glycans. In particular, some antibodies exhibit high specificity for unprocessed 'high mannose' type glycans, while others exhibit specificity for more highly processed 'complex type' glycans. A major gap in current knowledge is what types of glycans are present at each glycosylation site on gp120/gp41 on the intact virus. This is critical information for understanding the optimal epitopes of glycan-dependent bnAbs. In this project we seek to better define the epitopes of glycan-dependent bnAbs, and use the information to develop immunogens that elicit a neutralizing immune response that targets the glycan shield. We will use a novel glyco-proteomics approach to analyze and determine type of glycan (high mannose or complex) for each glycosylation site on gp120/gp41 from HIV viruses produced in laboratory cell lines and peripheral blood mononuclear cells. We will use this information to produce synthetic scaffolds that carry defined glycans for characterization of the optimal epitopes of bnAbs, and to generate recombinant gp120 immunogens that approximate the glycosylation of gp120 on intact virus. Both the synthetic scaffolds and glycosylation optimized recombinant gp120 trimers will be tested for induction of neutralizing immune responses mediated by glycan-dependent gp120 antibodies.